The objective of this proposal is to develop new probe targeting methodology based on the enzyme lipoic acid ligase (LplA). LplA catalyzes the site-specific covalent conjugation of the cofactor lipoic acid onto the lysine sidechain of one of three bacterial protein substrates. We propose to re-engineer LplA for probe targeting inside living mammalian cells. We previously engineered a transposable 12-amino acid peptide substrate for LplA, and mutagenized the lipoate binding pocket to accommodate a coumarin fluorophore. Here, we propose to (1) use a combination of rational design and directed evolution to engineer LplA-derived ligases for the red fluorophore resorufin and the photocrosslinker benzophenone. For directed evolution, we will explore FACS, cell laser ablation, and antibiotic selection strategies in parallel; (2) develop two-step probe targeting schemes based on LplA-catalyzed ligation of functional group handles such as alkyl azides; and (3) apply our new probe ligases to the study of neurexin and neuroligin biology in living neurons. Our work should benefit cell biologists interested in minimally- invasive analysis of protein function in single living cells, and neuroscientists interested in understanding the molecular bases of synapse development.